The relation between epidermal growth factor receptor mutations profiles and smoking patterns in patients with lung adenocarcinoma: A cross‐sectional study

Abstract Background Non‐small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, with smoking being a critical risk factor. The identification of NSCLC patients harboring epidermal growth factor receptor (EGFR) mutations, sensitized to tyrosine kinase inhibitors, has revolutionized treatment plans, resulting in improved clinical responses and reduced chemotherapy toxicity. This study aimed to assess the relationship between EGFR mutations and smoking patterns in patients diagnosed with lung adenocarcinoma referred to major pathologic laboratories. Methods This cross‐sectional study included 217 NSCLC patients aged above 18 years. Molecular abnormalities of the EGFR gene were analyzed by polymerase chain reaction amplification of exons 18–21 accompanied by Sanger sequencing. Then, the data were analyzed using the SPSS 26 software. Logistic regression analysis, χ 2 test, and Mann–Whitney U test were used to evaluate the relation between EGFR mutations and smoking patterns. Results EGFR mutations were identified in 25.3% of patients, predominantly involving deletion in exon 19 (61.8%). For most of the mutant EGFR patients, the majority were nonsmokers (81.8%), and 52.7% were female patients. Besides, the median duration of smoking was 26 years and the median frequency of smoking was 23 pack‐years in the mutant EGFR group, both of which were lower compared to the wild mutant group. Moreover, female gender, current, and heavy smoking were significantly correlated with EGFR mutations based on the univariate logistic regression analysis (p: 0.004, 0.005, and 0.001, respectively). Conclusions Female gender and nonsmoker status were strongly associated with positive EGFR mutations. While guidelines traditionally recommended EGFR testing primarily for female nonsmokers with advanced NSCLC, our study in line with the recently published evidence has shown a significant prevalence of positive EGFR mutations among male patients and smokers. Therefore, routine mutation testing is suggested for all NSCLC patients. Considering the limited access to EGFR testing laboratories in developing countries, the results of such epidemiological surveys can assist oncologists in choosing the most suitable treatment plan.

suggested for all NSCLC patients. Considering the limited access to EGFR testing laboratories in developing countries, the results of such epidemiological surveys can assist oncologists in choosing the most suitable treatment plan.

K E Y W O R D S
adenocarcinoma of the lung, cigarette smoking, EGFR gene, non-small cell lung cancer

| INTRODUCTION
Lung cancer (LC) is the leading cause of cancer deaths and the second most commonly diagnosed cancer worldwide. It is the primary cause of cancer-related deaths in males and the second in females. 1 According to recent global epidemiology data on LC that has been published recently, the highest frequency of LC was seen in Asian countries close to Eastern Europe such as Armenia, Turkey, and Kazakhstan. In contrast, Middle Eastern countries like Saudi Arabia and Yemen have reported the lowest frequency. 2 LC has been associated with multiple risk factors, including genetic, environmental, and occupational factors, such as active and passive smoking, various genomic changes, pre-existing lung diseases, and indoor and outdoor air pollution. [3][4][5] The incidence of LC varies due to different cigarette smoking patterns. 2 Studies conducted in our country have shown that smoking cigarettes is the most significant risk factor observed in approximately 60% of LC patients. 6,7 The related systematic review and meta-analyses have also shown a strong association between water-pipe smoking and the risk of LC progression. 8 Non-small cell lung cancer (NSCLC) is the most common type of LC, accounting for 85% of cases. NSCLC has two histological subtypes, that is, adenocarcinoma, which is the most prevalent histologic subtype in both sexes, 2,6 and squamous cell carcinoma.
Additionally, approximately one-third of NSCLC patients, especially those with adenocarcinoma, have been found to harbor epidermal growth factor receptor (EGFR) mutations. 9,10 EGFR mutations, the most common mutations observed in female and nonsmoker NSCLC patients, 5 lead to increase growth signal within the cell, eventually resulting in the development of cancer cells. 11 Recently, the recognition of NSCLC patients harboring EGFR mutations, who respond well to tyrosine kinase inhibitors (TKIs) like Gefitinib or Erlotinib, has revolutionized treatment plans, resulting in an improvement in clinical responses and quality of life. Therefore, first-line treatment with targeted therapies has shown superior clinical outcomes compared to conventional cytotoxic chemotherapy regimens in patients with EGFR mutations. 12,13 There is limited information regarding the epidemiology of EGFR mutations and smoking patterns in our region, and most surveys have not evaluated detailed smoking profiles and habits in EGFR-positive group. Hence, the current study aims to assess the relationship between EGFR mutations and smoking patterns among patients diagnosed with lung adenocarcinoma referred to pathological laboratories.

| Sample size measurements
On the basis of study by Wei et al., 14 considering the frequency of EGFR mutation as 21%, confidence interval (CI) of 95%, power of 21%, and d (margin of error) = 0.05, and using the n = Z 2 × P(1 − P)/ d 2 formula, 255 samples were determined for the study. A flow diagram illustrating the participant recruitment process is provided in Figure 1.
F I G U R E 1 Flow diagram of participant recruitment in the study. EGFR, epidermal growth factor receptor.

| Data collection and outcome measures
Demographic and pathological characteristics, including age, gender, EGFR mutation testing results, and phone number, were obtained from the laboratories. After a thorough explanation of the study's aim and obtaining consent to participate, the following information was collected over the phone from patients or they first-degree relatives Considering the cigarette smoking pattern, the patients were classified into three categories: • Current smokers (still smoking or quitting smoking less than 6 months before the disease diagnosis).
• Former smokers (quitting smoking more than 6 months before the disease diagnosis).
The smoking frequency was determined by multiplying the number of cigarette packs smoked per day by the number of smoking years.
Heavy smokers smoke greater than or equal to 20 or more cigarette packs a year, while light smokers are those who smoke less than 20 packs per year. The duration of the disease was measured from the onset of diagnosis by EGFR testing till the time of death or the time of the study. Polymerase chain reaction (PCR) amplification of exons 18-21 was done, followed by Sanger sequencing of the product. Although this test has high specificity, its sensitivity in detecting mutations is limited. The PCR amplification procedure was conducted in three stages within 2 h of execution. Primers used for sequencing the EGFR gene have been presented in Table 1 Table 1. All EGFR mutation test results were classified and analyzed in three categories, namely wild type, deletion in exon 19, and others. It should be noted that the patients did not undergo chemotherapy or radiotherapy before their surgical resection.

| EGFR mutation testing
This approach was taken to prevent any up or downregulation of cellcycle proteins caused by DNA damage.

| Statistical analysis
The data were presented as mean ± standard deviation (SD), median (interquartile range), and number (%). An Independent t test was employed to compare the EGFR-positive and EGFR-negative groups regarding age. A χ 2 test was used for categorical variables while the Mann-Whitney U test was utilized to evaluate the duration of disease, duration, and frequency of smoking which were nonparametric. Univariate and multivariate logistic regression analyses were conducted to identify predictors of EGFR mutations. The data were analyzed using the IBM Statistical Package for Social Sciences (SPSS, version 26; SPSS Inc.). p < 0.05 was considered statistically significant.
T A B L E 1 Primers used for sequencing of the epidermal growth factor receptor (EGFR) gene. Heavy smokers were also five times less prone to EGFR mutations than non-smokers (CI: 0.090-0.565, p: 0.001). However, the results from the multivariate logistic regression analysis, as summarized in Table 3, did not demonstrate any significant associations between the variables and EGFR mutations.

| DISCUSSION
The present study evaluated the incidence of EGFR mutations among smokers and nonsmokers with lung adenocarcinomas. Previous surveys have established a connection between adenocarcinoma histology and the high prevalence of EGFR gene mutations. 16 Estimation of the probability of EGFR mutations using common demographic information has been proposed as a valuable adjunct to laboratory testing especially in developing countries and regions without enough facility. 32 Information on the epidemiology of EGFR mutations and smoking patterns in our region is limited. The singlecenter study evaluated EGFR mutations in 103 Iranian NSCLC patients and showed 24% mutation frequency mostly point mutation on exon 21 and never smokers. 33 Another study with 50 patients reported EGFR-positive mutations in 28% of patients. Females, nonsmokers, and deletion in exon 19 were more common in the EGFR-positive group. 34

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

ETHICS STATEMENT
The manuscript has been approved by all authors and has never been published or under consideration for publication elsewhere. We confirm that all figures and tables are original and created by authors.
We guarantee that all authors listed on the title page have read the manuscript and attest to the validity and legitimacy of the data. We would also like to undertake that we have read the plagiarism policy and submitted the article with complete responsibility. This study was done in compliance with the Declaration of Helsinki and approved by the ethics committee of the university. All patients gave their informed consent before their inclusion in the study.

TRANSPARENCY STATEMENT
The lead author Alireza Rezvani affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.